Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension

Abstract

The aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPAinducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinasesdependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression.

Acidic Extracellular pH Induces Matrix Metalloproteinase-9 Expression in Mouse Metastatic Melanoma Cells through the Phospholipase D-Mitogen-activated Protein Kinase Signaling

“…Quantitative Reverse Transcription (RT)-PCR-MMP-9 mRNA expression was measured by quantitative RT-PCR using a synthetic RNA as the internal standard (37). Total RNA (10 ng) was extracted from cells using an Instra-Pure RNA purification kit, and the RNA was reverse-transcribed and amplified by a GeneAmp Thermostable rTth transcriptase RNA PCR kit.…”

Mechanoregulation of gene expression in fibroblasts

“…Expression levels of tenascin-C were found to be increased in chick embryo fibroblasts in restrained collagen gels (Chiquet-Ehrismann et al, 1994). In contrast, a group of MMPs (MMP-1, -3, -9, and -13) was upregulated in human dermal fibroblasts by dissipation of the tension upon retraction of collagen gel (Lambert et al, 2001). …”

Invasion and MMP expression profile in desmoid tumours

“…The mRNA of interest and the 28S rRNA were quantified by RT -PCR using 10 ng of total RNA. For each mRNA, the sequences of primers, the optimal conditions of reaction (number of amplification cycles and temperatures) and the amount of primers and internal standards were used as previously described Lambert et al, 2001), in order to be within the linear range of measurement under noncompetitive conditions. The efficiency of the RT and the amplification reactions was monitored by adding in each tube an appropriate number of copies of a synthetic RNA (sRNA) that can be reversetranscribed and amplified with the primer pair used for the endogenous mRNA of interest, but giving rise to a product slightly larger or smaller than the product amplified from mRNA, allowing its discrimination after migration in a 10% polyacrylamide gel.…”